Summary: The dengue virus (DEN) protease is a heterodimer of nonstructural protein 2B (NS2B) and NS3. Previously, we performed a mutagenesis study of some of the residues in NS3 of DEN type 2 (DEN2) predicted to be involved in substrate binding. In all, 46 mutations were analyzed for their effect on intramolecular cleavage at the NS2B-NS3 junction and intermolecular cleavage at the NS4B-NS5 junction in vitro. Thirteen of these mutants that had wild-type or nearly wild-type activity in vitro have now been analyzed by reverse engineering them into a full-length DEN2 cDNA clone and testing mutant RNA transcripts for infectivity: 5 were non-infectious, 7 were recovered as virus, and in 1 case the recovered virus was a pseudorevertant. The five lethal mutations had less activity in vitro, suggesting that DEN is not able to tolerate much reduction in protease activity. The recovered viruses appeared to have wild-type growth kinetics. In addition, work has begun to reverse engineer a set of previously made mutations in DEN4 NS2B into an infectious DEN4 clone made by Robin Levis. So far, 3 mutations have been crossed in, and at least one of the mutant RNAs was infectious. We had previously observed that transcripts made from DEN2 cDNA clones with short deletions at the 3' end are infectious. Constructs missing up to 6 nt are viable, while deletion of 8 or more nt is lethal. Deletions of 7 nt are viable or lethal, depending on the presence and composition of a restriction site overhang at the 3' end of the run-off transcript. In all but one case, the 3' end sequences of the recovered viruses are wild-type; virus recovered from the 7 nt deletion has a 3' end sequence that differs from wild-type at 1.5 positions, but this mutant virus appears to grow with wild-type kinetics. There is some evidence that the length and sequence composition at the 3' end of these deleted RNAs can rapidly evolve to wild-type, and RNAs with the wild-type sequence outgrows all the intermediate forms. In a collaboration with the Padmanabhan lab, a previously described set of chimeric DEN2-West Nile virus 3' end constructs were analyzed in their in vitro DEN RNA replication system. The in viro replication activity of these mutants closely paralleled their ability to replicate in cell culture. Finally, it has been universally found that full-length flavivirus clones are difficult to work with in E coli. Cells harboring the full-length clone are at a selective growth disadvantage compared to cells harboring clones with certain rearrangements of the flavivirus DNA, usually insertions or deletions. We have constructed a series of deletion and insertion mutants of the DEN2 clone to begin to characterize the mechanism for this instability.